Transgenic plant product development by conventional transformation and breeding efforts is a slow and unpredictable process. Gene targeting systems can overcome such problems as expression variability, unpredictable impacts of random gene insertion on agronomic performance, and the large number of experiments that need to be conducted to obtain ideal transgenic plants. Such systems can also provide approaches to manipulating endogenous genes.
Gene targeting systems require the ability to focus the recombination process to favor the recovery of desired targeting events. The natural cellular DNA repair and recombination machinery consists of a complex array of protein components interacting in a highly controlled manner to ensure that the fidelity of the genome is conserved throughout the many internal events or external stimuli experienced during each cell cycle. The ability to manipulate this machinery requires an understanding of how specific proteins are involved in the process, and how the genes that encode those proteins are regulated. Because many different protein components may be involved in gene targeting, the availability of host-specific genes and proteins could avoid possible problems of incompatibility associated with molecular interactions due to heterologous components.
The RAD2 gene of the budding yeast Saccharomyces cerevisiae is one of several genes known to be important in excision repair (1). It encodes an endonuclease that specifically cleaves single-stranded DNA in the 5′ to 3′ orientation. The yeast RAD2 gene and encoded protein (Rad2) exhibit high homology to a human DNA repair protein XP-G (2) and the structure specific Flap Endonuclease-1 or FEN-1 (3, 4). Human FEN-1 is a 380 amino acid protein which cleaves DNA flap strands that terminate with 5′ single strand ends. This cleavage is flap strand specific and independent of the flap strand sequence or length (3). Other branched structures such as Holliday junctions or double D loop are not cleaved by FEN-1 (3). Members of this family have also been cloned from the budding yeast (5), Xenopus (6), and mouse (7), as well as the archaebacteria (8). Recently, Kimura et al. have also characterized a structure-specific endonuclease from Brassica oleracea (9).
Genetic and biochemical studies have established that the Rad2/FEN-1 protein is a structure specific endonuclease (8, 10). Moreover, under certain reaction conditions, it also acts as an exonuclease (8, 10). The endonucleolytic activity is essential in DNA replication as well as the nucleotide excision repair reactions (8, 10). The exonucleolytic activity is involved in double strand break repair and end joining (8, 10). The protein is also useful in strand exchange reactions during homologous recombination (8, 10). These functions could prove to be very useful in gene targeting and in the production of male sterile plants. For example, the efficiency of gene targeting can be improved by the overexpression of exogenous Rad2/FEN-1 while male sterile plants can be produced by the down-regulation of Rad2/FEN-1 expression.
The regulation of DNA repair and recombination in plant systems by the modulation of maize Rad2/FEN-1 will provide improved and expanded methods of gene targeting. The need in the art for methods to regulate gene targeting and to modulate male sterility is clear. The present invention provides these and other advantages.